Due to environmental and regulatory concerns, research and development of non-chemical alternatives for pest suppression is necessary. Entomopathogenic nematodes are environmentally friendly biocontrol agents that can suppress a wide variety of economically important insect pests including the black vine weevil, the Japanese beetle, diaprepes root weevil, and fungus gnats. Commercialization of these nematodes, however, has not reached its potential. Our challenge is to overcome two critical barriers preventing widespread usage of entomopathogenic nematodes: 1) excessive costs in production, and 2) variable efficacy in field applications. Nematode production technology utilizing insect hosts is a well-suited approach for small businesses to produce quality nematode products. However, the costs of producing by this methods are high relative liquid fermentation approaches due to the expense of insect hosts and labor. Our objective is to determine the feasibility of fully mechanizing insect host mass production. The project will focus on producing nematodes in mealworm hosts. A basic system for producing these nematodes in mealworms is already in place, but the process lacks economy of scale and requires mechanization to achieve maximum efficiency. We propose developing a completely automated processes including production of improved quality insect host media, host inoculation, nematode harvest and packaging. OBJECTIVES: Overall Goal: to develop a fully mechanized in vivo production system for entomopathogenic nematodes using T. molitor as the host. Specific Objectives: 1. Optimize nutritional components of the T. molitor diet for maximum efficiency in nematode production. a. Determine the effect of diet on the nutritional composition of T. molitor by comparing chemical analyses of T. molitor larvae reared under different diets. b. Use the nutritional information from objective 1a to determine the potential to improve nematode quality and production by modifying T. molitor diet. 2. Mechanize production of T. molitor. a. Construct a prototype production system for mechanized host production. b. Compare the production efficiency of the prototype with current rearing technology. 3. Mechanize inoculation and harvest of H. bacteriophora and S. riobrave. a. Construct a prototype for inoculation and harvest of nematodes in cadavers and aqueous. b. Compare the production efficiency of the prototype with current rearing technology. 4. Mechanize packaging and storage of H. bacteriophora and S. riobrave. a. Construct a prototypes for mechanized packaging of nematodes in cadavers or aqueous. b. Determine longevity of and optimum densities in packaged material. 5. Determine quality of nematodes produced through the mechanized process. Compare quality (viability, virulence, and efficacy) of nematodes derived from objectives 1, 3 and 4 to nematodes produced using standard laboratory rearing procedures. 6. Conduct a preliminary analysis of the approach based on Phase I research. APPROACH: Obj. 1 - Determine the effect of diet on nutritional composition of mealworms by comparing chemical analysis of larvae reared on different diets. Focus will be on mealworm lipid content since components of lipids increase nematode yield and quality. We will demonstrate that mealworm hosts and nematode yield and quality can be improved through diet analysis and amendments to mealworm diet. Obj. 2 - Mealworm production is labor intensive work that requires separation of wheat bran and/or feces from various life forms of mealworms (ie., adults from eggs and bran; larvae from bran; larvae from feces; large larvae from small larvae; larvae from pupae). We will construct a prototype electrically operated sifter having a series of stacked screens of appropriate mesh sizes. Conveyor belts will load and unload the sifter and discharge life forms, bran and feces into appropriate trays or bins. Tray forming equipment is planned for producing disposable rearing trays to replace permanent trays. Obj. 3 - Inoculation of mealworms to produce cadavers or aqueous nematodes for sale is very labor intensive. We will construct a prototype device for infecting very large numbers of mealworm larvae. The prototype consists of large plaster of Paris shelves stacked within a temperature and humidity controlled cabinet. Inoculum will be pumped onto each shelf where the plaster will absorb the water leaving nematodes ready to infect mealworms. Mealworms will be volumetrically metered onto each shelf. Three days later infected mealworms will be transferred to one of several prototype devices designed for packaging cadavers or for harvesting nematodes for use in aqueous application. Use and development of the prototypes are described in the following objective. Obj. 4 - The prototype for packaging cadavers automatically spaces cadavers onto adhesive face tape. The tape and cadavers is then covered with fabric to hold cadaver placement. Tapes are then formed into rolls and placed in resealable pouches for shipment to users. The prototype for harvesting nematodes is an aerated tank containing water above which is located a fabric shelf to support the cadavers until they emerge into the water. Once emerged, the nematodes are quantified and then pumped into an aerated-refrigerated holding tank for concentrating or packaging for sale. Obj. 5 - Quality of nematodes produced by Obj. 1, 3 and 4 will be compared for viability, virulence, and efficacy using standard laboratory procedures. Citrus root weevils and black vine weevils will be used to assess virulence. Obj. 6 - Treatment effects in all experiments will be analyzed using standard statistical procedure. Based on data collected during Phase I we will evaluate the overall mechanized approach by weighing projected efficiency and cost of nematode production efficiency
